Vector Identification and Gene Delivery Approach in Pigs

描述(由申请人提供)： 这项建议的目的是开发一种新的治疗分子变力作用的方法，特别是通过病毒介导的钙调节蛋白SERCA 2a(肌浆网钙ATPase)传递给表现为心力衰竭的大动物，试图在不恶化能量参数的情况下恢复功能和提高存活率。充血性心力衰竭(CHF)是一个巨大的临床问题，需要有效的治疗方法。尽管CHF的传统治疗方法取得了进展，包括药物治疗、心肌血管重建术、机械辅助装置和移植，但CHF仍然是全球主要的死亡原因。因此，旨在降低CHF发病率和死亡率、提高数百万患者生活质量的新型治疗方法就显得尤为诱人。心脏基因治疗在早期的动物研究中显示出了希望，并有助于心力衰竭的治疗。细胞内钙处理的缺陷被认为是人类和实验性充血性心力衰竭的关键异常。在人和动物心力衰竭模型中，肌浆网(SR)在松弛过程中的钙摄取不足与肌浆网钙离子ATPase(SERCA2a)的表达和活性降低有关。我们在过去五年中的初步工作表明，1)基因转移是将SERCA2a基因导入体内外心肌细胞的有效手段；2)增加SERCA2a基因的表达可以恢复CHF啮齿动物模型的收缩能力，并使细胞内钙循环正常化。我们现在正在将我们的实验从啮齿动物扩展到猪模型。我们的目标是1)确定病毒载体在分离的猪心心肌细胞中的转导效率；2)确定AAV载体和E1-E4缺失的重组腺病毒在猪体内基因转移后的转导效率；3)检测近端的人脑钠素(HBNP)启动子(相对于转录起点-408到+100)对压力超负荷和缺血诱导的能力。

DESCRIPTION (provided by applicant): The goal of this proposal is to develop a novel therapy for molecular inotropy, specifically, viral-mediated myocardial delivery of the calcium regulatory protein SERCA 2a (sarcoplasmic reticulum Ca 2¿ATPase) to large animals exhibiting heart failure in an attempt to restore function and improve survival without worsening energetic parameters. Congestive heart failure (CHF) represents an enormous clinical problem demanding effective therapeutic approaches. Despite advances in traditional approaches to its treatment, including pharmacologic management, myocardial revascularization, mechanical assist devices, and transplantation, CHF remains a leading cause of death worldwide. Therefore, a novel therapy aimed at decreasing the morbidity and mortality of CHF and improving the quality of life for millions of patients is particularly attractive. Cardiac gene therapy has shown promise in early animal studies and lends itself to the treatment of heart failure. A defect in intracellular calcium handling is known to be a key abnormality in both human and experimental CHF. Deficient Ca 2¿uptake by the sarcoplasmic reticulum (SR) during relaxation in failing hearts from humans and animal models has been associated with a decrease in the expression and activity of SR Ca2+ATPase (SERCA2a). Our preliminary work over the last five years have shown that 1) Gene transfer is an effective means of introducing the SERCA2a gene into myocytes in vitro and in vivo and 2) that increasing the expression of SERCA2a restores contractility and normalizes intracellular calcium cycling in a rodent model of CHF. We are now extending our experiments from rodents to porcine models. We aim to 1) determine the efficiency of transduction of viral vectors in cardiomyocytes isolated from pig hearts; 2) determine the efficiency of transduction of the AAV vectors and El-E4 deleted recombinant adenovirus following gene transfer in vivo in pigs; 3) test the proximal human brain natriuretic (hBNP) promoter (-408 to +100 relative to transcription start site) for the ability to be induced by pressure-overload versus ischemia.